Hydogen peroxide-dependent photocytotoxicity by phloxine B,a xanthene-type food colorant

Background

Phloxine B (PhB; 2′,4′,5′,7′-tetrabromo-4,5,6,7-tetrachloro-fluorescein), an artificial xanthene colorant, has been used as a red coloring agent in drugs and cosmetics as well as foods in some countries. However, little effort has been devoted to the study of this colorant as a potentially useful medicinal agent.

Methods

We investigated the daily light-induced photocytotoxicity of PhB in two human leukemia cells, HL-60 and Jurkat, and its underlying mechanisms by in vitro experiments using antioxidants.

Reuslts and conclusions

PhB inhibited cell proliferation more preferentially to HL-60 cells than to Jurkat cells. Co-treatment of catalase completely blocked the photocytotoxicity by PhB in HL-60 cells, whereas the effect of histidine was only partial, suggesting that hydrogen peroxide (H₂O₂), rather than singlet oxygen, might be a prerequisite for the PhB-induced HL-60 cell death. Actually, PhB produced a significant amount of H₂O₂ in the media as well as in the cells in concentration- and light-dependent manners. Furthermore, methionine, a hypochlorous acid (HOCl) scavenger, also significantly attenuated the cytotoxicity in HL-60 cells, but not in Jurkat cells, indicating the involvement of myeloperoxidase (MPO)-dependent hypohalous acid formation during the photocytotoxicity. In vitro experiments revealed that halogenated tyrosine was generated from the reaction of bovine serum albumin with PhB and HL-60 cell lysate. The present findings suggested that PhB induced a differential photodynamic action in the MPO-containing leukemia cells through an H₂O₂-dependent mechanism.

General significance

Our findings provide new insights into the molecular mechanisms underlying the PhB-induced apoptosis and also evaluated PhB as a promising PDT agent.     

Effect of auxin and other hormones on the metabolic response to wounding in sweet potato roots

In roots of sweet potato (Ipomoea batatas Lam. cv. Kokei 14),the metabolic response to wounding was remarkable only in theproximal side. We assumed that the polarity resulted from apolar movement of indole-3-acetic acid (IAA) produced in thecut surface (8). As the metabolic response was slight in thedistal side, the effect of IAA and the other plant hormoneson the development of various enzyme activities was examinedin this side. Increases in activities of L-phenylalanine ammonia-lyase,acid invertase, NADPHa₂ : cytochrome c oxidoreductase, peroxidase,cytochrome c : O₂ oxidoreductase and o-diphenol oxidase, whichdeveloped in response to wounding, were stimulated by the treatmentwith IAA. Gibberellic acid had a stimulative effect on the developmentof only acid invertase activity. Abscisic acid and kinetin hadlittle effect. The results strongly support our hypothesis thatIAA plays an important role in the metabolic response to wounding. (Received September 29, 1979; )     

Action mechanism of tachyplesin I and effects of PEGylation

PEGylation of protein and peptide drugs is frequently used to improve in vivo efficacy. We investigated the action mechanism of tachyplesin I, a membrane-acting cyclic antimicrobial peptide from Tachypleus tridentatus and the effects of PEGylation on the mechanism. The PEGylated peptide induced the leakage of calcein from egg yolk l-α-phosphatidylglycerol/egg yolk l-α-phosphatidylcholine large unilamellar vesicles similarly to the parent peptide. Both peptides induced lipid flip-flop coupled to leakage and was translocated into the inner leaflet of the bilayer, indicating that tachyplesin I forms a toroidal pore and that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. Despite their similar activities against model membranes, the peptides showed very different biological activities. The cytotoxicity of tachyplesin I was greatly reduced by PEGylation, although the antimicrobial activity was significantly weakened. We investigated the enhancement of the permeability of inner membranes induced by the peptides. Our results suggested that outer membranes and peptidoglycan layers play an inhibitory role in the permeation of the PEG moiety. Furthermore, a reduction in DNA binding by PEGylation may also contribute to the weak activity of the PEGylated peptide.     

Growth factor contents of autologous human sera prepared by different production methods and their biological effects on chondrocytes

To discuss the autologous serum production for cartilage tissue engineering, we compared three kinds of sera: whole blood-derived serum (WBS), platelet-containing plasma-derived serum (PCS), and plasma-derived serum (PDS), on the growth factor contents and their biological effects on human auricular chondrocytes. EGF, VEGF and PDGF levels were highest in WBS, while PCS and PDS followed WBS. The proliferation effects of WBS were the most pronounced, followed by that of PCS, both of which realized a 1000-fold-increase in chondrocyte numbers at the third passage, whereas PDS reached it after passage 4. No significant differences were observed in histology or cartilaginous matrix measurements of tissue-engineered cartilage produced from chondrocytes cultured under different serum conditions. WBS would be clinically useful because of its potent proliferation effects, while PCS, which possibly saves the red cell concentrate, may be an option in cases where there are elevated risks of blood loss.     

Liposome Vector Containing Biosurfactant-Complexed DNA as Herpes Simplex Virus Thymidine Kinase Gene Delivery System

For injectable-sized liposome complexed with DNA (lipoplexes) with high transfection efficiency of genes, we initially prepared small-sized liposomes by addition of biosurfactant. For selectivity of gene expression, the thymidine kinase (MK-tk) gene controlled by midkine was used for herpes simplex virus thymidine kinase (HSV-tk) gene therapy. Liposomes composed of 3([N-(N′,N′–dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), L-dioleoylphosphatidylethanolamine (DOPE), and a biosurfactant, such as β-sitosterol β-D-glucoside (Sit-G) for Sit-G-liposomes and mannosylerythrytol lipid A (MEL) for MEL-liposomes, produced about 300-nm-sized lipoplexes. Sit-G- and MEL-liposomes showed higher transfection efficiency of the luciferase marker gene and thymidine kinase activity in the presence of serum in the cells. The treatment with transfection of MK-tk gene by Sit-G-liposome and injection of ganciclovir significantly reduced tumor growth in a solid tumor model, compared with that by Sit-G-liposome alone. This finding suggested that Sit-G-liposome is a potential vector for HSV-tk gene therapy.     

Development of α1,6-fucosyltransferase inhibitors through the diversity-oriented syntheses of GDP-fucose mimics using the coupling between alkyne and sulfonyl azide

We developed α1,6-fucosyltransferase (FUT8) inhibitors through a diversity-oriented synthesis. The coupling reaction between the fucose unit containing alkyne and the guanine unit containing sulfonyl azide under various conditions afforded a series of Guanosine 5′-diphospho-β-l-fucose (GDP-fucose) analogs. The synthesized compounds displayed FUT8 inhibition activity. A docking study revealed that the binding mode of the inhibitor synthesized with FUT8 was similar to that of GDP-fucose.     

(−)‐Epigallocatechin‐3‐gallate inhibits human angiotensin‐converting enzyme activity through an autoxidation‐dependent mechanism

We investigated the molecular mechanisms involved in the angiotensin‐converting enzyme (ACE) inhibition by (?)‐epigallocatechin‐3‐gallate (EGCg), a major tea catechin. EGCg inhibited both the ACE activity in the lysate of human colorectal cancer cells and human recombinant ACE (rh‐ACE) in a dose‐dependent manner. Co‐incubation with zinc sulfate showed no influence on the rh‐ACE inhibition by EGCg, whereas it completely counteracted the inhibitory effect of ethylenediaminetetraacetic acid, a chelating‐type ACE inhibitor. Although hydrogen peroxide was produced by the autoxidation of EGCg, hydrogen peroxide itself had little effect on the ACE activity. Conversely, the co‐incubation of EGCg with borate or ascorbic acid significantly diminished the EGCg inhibition. A redox‐cycling staining experiment revealed that rh‐ACE was covalently modified by EGCg. A Lineweaver–Burk plot analysis indicated that EGCg inhibited the ACE activity in a non‐competitive manner. These results suggested that EGCg might allosterically inhibit the ACE activity through oxidative conversion into an electrophilic quinone.     

Correlations of calcium accumulations in arteries, veins, cartilages, ligaments, and bones in single humans

To show the relationships of calcium accumulation in the thoracic aorta to the other tissues, calcium contents were determined with a microwave-induced plasma-atomic emission spectrometer on arteries, veins, cartilages, ligaments, and bones. These tissues were resected from 18 individuals, consisting of 11 men and 7 women who died in the age range 59–91 yr. As thoracic and abdominal aortas are routinely used for radiographic examination of arterial calcification, they appear to be standard tissues of the calcium accumulation. The calcium accumulations were determined in the femoral artery, the superior and inferior venae cavae, the internal jugular vein, cartilages of the articular disk of the temporomandibular joint and the intervertebral disk, both the ligaments of the anterior cruciate ligament and the ligamentum capitis femoris, and the calcaneus, in contrast with the thoracic aorta. As calcium increased in the thoracic aorta, it increased in the femoral artery, the articular disk of the temporomandibular joint, the intervertebral disk, both ligaments of the anterior cruciate ligament, and the ligamentum capitis femoris, but it did not increase in veins, such as the superior and inferior venae cavae and the internal jugular vein. In contrast, it decreased in the calcaneus.     

Extraction of leukemia specific glycan motifs in humans by computational glycomics

There have been almost no standard methods for conducting computational analyses on glycan structures in comparison to DNA and proteins. In this paper, we present a novel method for extracting functional motifs from glycan structures using the KEGG/GLYCAN database. First, we developed a new similarity measure for comparing glycan structures taking into account the characteristic mechanisms of glycan biosynthesis, and we tested its ability to classify glycans of different blood components in the framework of support vector machines (SVMs). The results show that our method can successfully classify glycans from four types of human blood components: leukemic cells, erythrocyte, serum, and plasma. Next, we extracted characteristic functional motifs of glycans considered to be specific to each blood component. We predicted the substructure alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-D-GlcpNAc as a leukemia specific glycan motif. Based on the fact that the Agrocybe cylindracea galectin (ACG) specifically binds to the same substructure, we conducted an experiment using cell agglutination assay and confirmed that this fungal lectin specifically recognized human leukemic cells.